This invention relates to using a pectic substance to stabilize a pectin/heparin-binding protein, the resultant composition and formulation, and the method of stabilizing the pectin/heparin-binding protein.
Following abbreviations are used in this invention:
aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; BSA, bovine serum albumin; Da, dalton; DAc, degree of acetylation; DM, degree of methylation; EDTA, ethylenediaminetetraacetic acid; EGF, epidermal growth factor; Gal A, galacturonic acid; HM, high methoxyl; HMW, high-molecular-weight; IL, interleukin; kDa, kilodalton; KGF, keratinocyte growth factor; LM, low methoxyl; LM, low-molecular-weight; PDGF, platelet-derived growth factor; SDS, sodium dodecyl sulfate; TGF-.alpha., transforming growth factor-.alpha.; TGF-.beta.1, transforming growth factor-.beta.1; TN buffer, 25 mM Tris, 0.15M NaCl, pH 7.4; TNF-.alpha., tumor necrosis factor-.alpha..
Pectin is a plant cell wall component. The cell wall is divided into three layers, middle lamella, primary, and secondary cell wall. The middle lamella is the richest in pectin. Pectins are produced and deposited during cell wall growth and are particularly abundant in soft plant tissues under conditions of fast growth and high moisture content. In cell walls, pectins are present in the form of a calcium complex. The involvement of calcium cross-linking is substantiated by the fact that chelating agents facilitate the release of pectin from cell walls.
Pectin is a complex polysaccharide. It consists of an .alpha.1-4 linked polygalacturonic acid backbone intervened by rhamnose residues and modified with neutral sugar side chains and non-sugar components such as acetyl, methyl, and ferulic acid groups. The neutral sugar side chains which include arabinan and arabinogalactans (Types I and II) are attached to the rhamnose residues in the backbone at the O-3 or O-4 position. The rhamnose residues tend to cluster together on the backbone. So with the side chains attached this region is referred as the hairy region and the rest of the backbone is hence named the smooth region. Rhamnose residues are 1-2 linked to Gal A residues in the backbone and the configuration of this linkage has now been determined to be .alpha..
Because of the presence of neutral sugar side chains and some other non-sugar components, the structure of pectins is very complex; essentially no two molecules have identical structures.
Rhamnose, galactose, arabinose, and xylose are the most common neutral sugar components of pectins. The less common ones are glucose, mannose, and fucose. Some of the xylose residues are individually attached to Gal A residues at the O-3 position. Three types of neutral sugar side chains have been identified in pectins. Arabinan consists of .alpha.1-5 linked arabinose. Arabinogalactan I consists of .beta.1-4 linked galactose with short arabinan chains attached at O-3. In arabinogalactan II, galactose is .beta.1-3&6 linked with arabinose attached.
Methylation occurs at carboxyl groups of Gal A residues. The degree of methyl-esterification is defined as the percentage of carboxyl groups (Gal A residues) esterified with methanol. A pectin with a degree of methylation ("DM") above 50% is considered a high methoxyl ("HM") pectin and one with a DM&lt;50% is referred to as low methoxyl ("LM") pectin. Most of the natural pectins are HM with a few exceptions such as sunflower pectin. The degree of acetylation (DAc) is defined as the percentage of Gal A residues esterified with one acetyl group. It is assumed that only the hydroxyl groups are acetylated. Since each Gal A residue has more than one hydroxyl group, the DAc can be above 100%. DAc is generally low in native pectins except for some such as sugar beet pectin.
Generally, pectins are soluble in water and insoluble in most organic solvents. Pectins with a very low level of methyl-esterification and pectic acids are only soluble as the potassium or sodium salts. As for other polymers, there is no saturation limit for pectins, but it is difficult to obtain a true solution with concentrations higher than 3-4%. Commercial pectins have a size range of 7-14.times.10.sup.4 Da with citrus pectins being larger than apple pectins. Viscosities of pectin solutions are generally low and so pectins are seldom used as thickening agents. The viscosity is directly related to the size, pH, and also to the presence of counterions. Addition of monovalent cations reduces viscosity.
The Gal A residues in the pectin backbone are .alpha.1-4 linked. Both hydroxyl groups of D-Gal A at carbon atoms 1 and 4 are in the axial position. The resulting linkage is therefore trans 1-4. This type of linkage results in increased chain stiffness of the polymer. So pectin with a flexibility parameter B between 0.072-0.017 are rigid molecules. It has been suggested that the insertion of rhamnose residues in the backbone cause a T-shaped kink in the backbone chain. An increase in rhamnose content leads to more flexible molecules. Pectins can be considered as a zigzag polymer with long and rigid smooth regions and flexible hairy regions (rich in rhamnose) serving as rotating joints. The DM also has certain effects on chain flexibility. In solution, pectin molecules have been shown to assume a right-handed helical structure.
Pectins are most stable at pH 3-4. Below pH 3, methoxyl and acetyl groups and neutral sugar side chains are removed. At elevated temperatures, these reactions are accelerated and cleavage of glycosidic bonds in the galacturonan backbone occurs. Under neutral and alkaline conditions, methyl ester groups are saponified and the polygalacturonan backbone breaks through .beta.-elimination-cleavage of glycosidic bonds at the non-reducing ends of methoxylated galacturonic acid residues. These reactions also proceed faster with increasing temperature. Pectic acids and LM pectins are resistant to neutral and alkaline conditions since there are no or only limited numbers of methyl ester groups.
Both HM and LM pectins can form gels, but by totally different mechanisms. HM pectins form gels in the presence of high concentrations of co-solutes (sucrose) at low pH. LM pectins form gels in the presence of calcium. In addition, the sugar beet pectin can form gels through cross-linking of the ferulated groups.
The calcium-LM pectin gel network is built by formation of the "egg-box" junction zones in which Ca++ ions cause the cross-linking of two stretches of polygalacturonic acids. In apple and citrus pectins, stretches of polygalacturonic acids without rhamnose insertion have been estimated to be as long as 72-100 residues. The zone is terminated by the rhamnose residue in the backbone. The calcium-LM pectin gel is thermoreversible. The calcium can therefore be added at the boiling point and gel formation occurs upon cooling. It is possible to obtain a firm resilient gel with 0.5% pectin and 30-60 mg/g Ca++. A high content of pectin with little calcium gives an elastic gel; whereas, a high calcium concentration with a minimum of pectin results in a brittle gel.
Industrial pectins, either HM or LM, are mainly obtained from apple and citrus by acid extraction and alcohol precipitation. LM pectins are obtained from HM ones by chemical de-esterification. Pectins have a favorable regulatory status as a food additive. They are classified as Generally Recognized As Safe ("GRAS") in the United States and Acceptable Daily Intake ("ADI") in Europe. That is, its use is only limited by current Good Manufacturing Practice ("cGMP") requirements to meet certain specifications. These specifications include a minimal Gal A content of 65% (w/w).
Many other plant sources have also been examined for pectin production. Two of them, sugar beet pulp and sunflower head, have been studied extensively. Both are abundant as raw materials. However, sugar beet pectin has a poor gel forming ability largely due to its high acetyl group content and small molecular size (.about.5.times.10.sup.4 Da). The sunflower pectins are naturally LM and can be efficiently extracted with chelating agents. Chemically, sunflower head pectin has a very high Gal A content and is a natural LM pectin, whereas sugar beet pectin has a relatively low Gal A content and a very high content of acetyl and ferulic acid groups. The structures of apple and citrus pectins are very similar to each other. Pectins are traditionally used as food additives. However, their use has extended into pharmaceutical areas as well. They often suffer from poor quality of raw materials and poor color quality (usually tan) of the pectin end products.
Pectins from different plant sources have different characteristics. In general, all commercial pectins including those that have gone through further processing have a certain degree of coloration as a final product. The color ranges from light yellow/brown (citrus pectin) to dark tan (apple and sunflower head pectins). The coloration is caused by the combination of two factors: natural color (pigmentation) of the raw materials and their content of polyphenols.
Pectins are effective against gastrointestinal ulcers and enterocolitis. Pectins also influence cell proliferation in the intestines. They also have a blood cholesterol-lowering effect and exhibit inhibition of antherosclerosis. This effect is the result of interactions between pectins and bile salts. Pectins have also been shown to affect the fibrin network in hypercholesterolaemic individuals. Pectins are also effective in removing lead and mercury from the digestive tract and repiratory organs.
Proteins possess unique chemical and physical properties which pose difficult stability problems: a variety of degradation pathways exist of proteins, involving both chemical and physical instability. These macromolecules are also at risk from microbial degradation due to adventitious contamination of the solutions during purification or storage. All these considerations are especially critical for the pharmaceutical manufacturer who is formulating and packaging these agents. Thus, a thorough pre-formulation program is an essential step for protein drugs, to solve their possible formulation problems.